Antibodies that bind il-23

ABSTRACT

The present invention provides an antibody that binds to the p19 subunit of human IL-23 and is characterized as having high affinity, selective, and neutralizing properties. The antibody is useful in the treatment or prevention of an autoimmune or inflammatory condition selected from the group consisting of consisting of multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases, ankylosing spondylitis, graft-versus-host disease, lupus and metabolic syndrome. The antibody is also useful in the treatment of cancer.

The present invention relates to antibodies that bind humaninterleukin-23 (IL-23) and uses thereof.

Interleukin-23 (IL-23) is a disulfide linked heterodimeric cytokinecomposed of a p19 and p40 subunit. It is part of the interleukin-12(IL-12) family of cytokines. IL-12 is a heterodimeric cytokine of 70 kDaconsisting of covalently linked p40 and p35 subunits. IL-12 plays acritical role in the development of protective innate and adaptiveimmune responses and in tumour surveillance. IL-12 has also beenimplicated in the inflammatory response through its capacity to promoteT helper type 1 (Th1) responses. However, the functional role of IL-12in the inflammatory response has been re-evaluated with the discovery ofthe related cytokine, IL-23. IL-23 is composed of the same p40 subunitas IL-12 but is covalently paired with a p19 subunit. Many of thereagents used to assess the role of IL-12 are directed against theshared IL-12/IL-23 p40 subunit, meaning that the activities previouslyascribed to IL-12 may have been mediated via IL-23. The development ofIL-23 deficient mice enabled investigators to distinguish between theactivities of IL-12 and IL-23 and identified IL-23 as an essentialmediator of the autoimmune/inflammatory response.

The functional IL-23 receptor is a heterodimer of the IL-12Rβ1 subunit,which is shared with the IL-12 receptor, and an IL-23R subunit. Thereceptor for IL-23 is constitutively associated with Janus kinase 2(Jak2) and predominantly activates STAT3, with less STAT4 activationthan IL-12.

The IL-23 receptor is expressed on activated/memory T-cells and naturalkiller (NK) cells. Monocytes, macrophages and dendritic cells alsoexpress IL-23 receptor at low levels. IL-23 supports the differentiationand maintenance of naive CD4+ T-cells into a novel subset of cellscalled Th17 cells, which are distinct from the classical Th1 and Th2cells. Th17 cells produce interleukin-17A (IL-17A) and interleukin-17F(IL-17F). Th17 cells produce a range of other factors known to driveinflammatory responses, including tumor necrosis factors known to driveinflammatory responses, including tumor necrosis factor alpha (TNF-α),interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor(GM-CSF), CXCL1 and CCL20. NK cells and innate lymphoid cells such aslymphoid tissue induce (LTi)-like cells express IL-23 receptor andretinoic-acid-related orphan receptor (ROR) gamma and produce IL-17 inresponse to IL-23. IL-1β and IL-23 also co-stimulate gamma-delta T cellsto induce IL-17 production without T cell receptor engagement.

There is substantial evidence that IL-23 responsive cells are associatedwith autoimmune inflammatory diseases and cancer. In particular, anIL-23 specific inhibitor (i.e. an inhibitor that inhibits IL-23 but notIL-12) would be particularly useful as inhibiting IL-23 withoutaffecting IL-12 is hypothesized to maximize therapeutic benefit whileminimizing the risk of suppression of host defenses.

Antibodies that specifically bind to the p19 subunit of IL-23 arepotentially useful inhibitors, see, for example, WO 2007/024846 and WO2007/027714. A problem with the antibodies disclosed in WO 2007/024846,at least, is the potential for tissue cross-reactivity, in particular,the potential to bind retinal tissue, which is a safety concern.Furthermore, the antibodies disclosed in WO 2007/024846, at least, havesub-optimal physical-chemical properties, for example, extremehydrophobicity leading to aggregation, that present a significantbarrier to production of the antibodies on an industrial scale.Additionally, no antibody targeting the p19 subunit of IL-23 has beenapproved for therapeutic use.

Thus, there remains a need for IL-23 antibodies. In particular, thereremains a need for IL-23 antibodies that bind with high affinity to thep19 subunit of IL-23, in particular, human IL-23, and do not bind to thep40 subunit of the related cytokine family member, IL-12. Moreparticularly, there remains a need for IL-23 antibodies that bind withhigh affinity to the p19 subunit of IL-23 and do not observably exhibittissue cross-reactivity, in particular, retinal tissue cross-reactivity.There is also a need for IL-23 antibodies that possess pharmaceuticallyacceptable physical-chemical properties that facilitate development,manufacturing or formulation.

The present invention provides an antibody that binds to the p19 subunitof human IL-23 comprising a light chain and a heavy chain, wherein thelight chain comprises a light chain variable region (LCVR) and the heavychain comprises a heavy chain variable region (HCVR), wherein the LCVRcomprises amino acid sequences LCDR1, LCDR2, and LCDR3, and the HCVRcomprises amino acid sequences HCDR1, HCDR2, and HCDR3, wherein LCDR1 isSEQ ID NO:4, LCDR2 is SEQ ID NO:5, LCDR3 is SEQ ID NO:6, HCDR1 is SEQ IDNO:1, HCDR2 is SEQ ID NO:2, and HCDR3 is SEQ ID NO:3.

In an embodiment of the present invention, the antibody comprises alight chain and a heavy chain, wherein the light chain comprises a lightchain variable region (LCVR) and the heavy chain comprises a heavy chainvariable region (HCVR), wherein the amino acid sequence of the LCVR isSEQ ID NO: 8 and the amino acid sequence of the HCVR is SEQ ID NO: 7.

In a further embodiment of the present invention, the antibody comprisestwo light chain variable regions (LCVRs) and two heavy chain variableregions (HCVRs), wherein the amino acid sequence of each LCVR is SEQ IDNO: 8 and the amino acid sequence of each HCVR is SEQ ID NO: 7.

In a still further embodiment of the present invention, the antibodycomprises a light chain and a heavy chain, wherein the amino acidsequence of the light chain is SEQ ID NO: 10 and the amino acid sequenceof the heavy chain is SEQ ID NO: 9.

In a still further embodiment of the present invention, the antibodycomprises two light chains and two heavy chains, wherein the amino acidsequence of each light chain is SEQ ID NO: 10 and the amino acidsequence of each heavy chain is SEQ ID NO: 9.

The present invention provides an antibody that binds to the p19 subunitof human IL-23 comprising a light chain and a heavy chain wherein thelight chain comprises a light chain variable region (LCVR) and the heavychain comprises a heavy chain variable region (HCVR), wherein the LCVRcomprises complementarity determining regions LCDR1, LCDR2, and LCDR3,and the HCVR comprises complementarity determining regions HCDR1, HCDR2,and HCDR3, and wherein LCDR1 consists of amino acid sequence SEQ IDNO:4, LCDR2 consists of amino acid sequence SEQ ID NO:5, LCDR3 consistsof amino acid sequence SEQ ID NO:6, HCDR1 consists of amino acidsequence SEQ ID NO:1, HCDR2 consists of amino acid sequence SEQ ID NO:2,and HCDR3 consists of amino acid sequence SEQ ID NO:3.

The present invention also provides an antibody that binds to the p19subunit of human IL-23 comprising a light chain and a heavy chain,wherein the light chain comprises a light chain variable region (LCVR)and the heavy chain comprises a heavy chain variable region (HCVR),wherein the LCVR chain comprises amino acid sequence SEQ ID NO: 8 andthe HCVR chain comprises amino acid sequence SEQ ID NO: 7.

The present invention also provides an antibody that binds to the p19subunit of human IL-23 comprising a light chain and a heavy chain,wherein the light chain comprises amino acid sequence SEQ ID NO: 10 andthe heavy chain comprises amino acid sequence SEQ ID NO: 9.

The present invention also provides an antibody that binds to the p19subunit of human IL-23 comprising two light chains and two heavy chains,wherein each light chain comprises amino acid sequence SEQ ID NO: 10 andeach heavy chain comprises amino acid sequence SEQ ID NO: 9.

The amino acid sequences of the antibodies of the present invention areprovided below.

SEQ ID NOs Heavy Light Antibody Chain Chain HCVR LCVR I 9 10 7 8Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 I 1 2 3 4 5 6

The present invention also provides an antibody that binds to the p19subunit of human IL-23 at a conformational epitope within amino acidpositions 81-99 and 115-140 of SEQ ID NO: 15.

The present invention also provides an antibody that binds to the p19subunit of human IL-23 at a conformational epitope within amino acidpositions 81-99 and 115-140 of SEQ ID NO: 15, wherein the antibodycontacts at least amino acid residues 94P, 95S, 97L, 98P, 99D, 123W,130S, 133P and 137W of SEQ ID NO: 15.

In a still further embodiment of the present invention, the antibody isselective to the p19 subunit of human IL-23.

When bound to the p19 subunit of human IL-23, the antibody of thepresent invention prevents binding of human IL-23 to the IL-23 subunitof the IL-23 receptor. Accordingly, the antibody of the presentinvention inhibits the activity of human IL-23 at the human IL-23subunit of the IL-23 receptor.

The antibody of the present invention does not prevent binding of humanIL-23 to the IL-12Rβ1 subunit of the IL-23 receptor and, therefore, doesnot inhibit the activity of human IL-23 at the IL-12Rβ1 subunit of theIL-23 receptor.

The antibody does not detectably bind to the p40 subunit shared by humanIL-23 and human IL-12.

In a still further embodiment of the present invention, the antibody theantibody has neutralizing activity to the p19 subunit of human IL-23.

In a still further embodiment of the present invention, the antibody ofthe present invention has an IC₅₀ of less than or equal to about 90 pM.Preferably, the antibody of the present invention has an IC₅₀ of lessthan or equal to about 74 pM. The IC₅₀ values are measured in an invitro murine splenocyte assay as described in the section entitled “InVitro Neutralization of Human or Cynomolgus Monkey IL-23 by Antibody Iin Murine Splenocytes” in Example 1.

In a still further embodiment of the present invention, the antibody ofthe present invention is selective and has neutralizing activity to thep19 subunit of human IL-23.

In a still further embodiment of the present invention, the antibody ofthe present invention has a dissociation equilibrium constant, K_(D), ofabout 10 pM to about 30 pM for human IL-23. Preferably, the antibody ofthe present invention has a K_(D) of about 21 pM for human IL-23. TheK_(D) values are established by binding kinetics at 37° C. as describedin the section entitled “Affinity Binding Measurement by Surface PlasmonResonance (BIAcore for Antibody I” in Example 1. The antibody of thepresent invention is further characterized with a k_(on) rate to the p19subunit of human IL-23 of from about 2.2×10⁶ M⁻¹sec⁻¹ to about 2.6×10⁶M⁻¹sec⁻¹. Preferably, the antibody of the present invention has a k_(on)rate to the p19 subunit of human IL-23 of about 2.43×10⁶ M⁻¹sec⁻¹. Theantibody of the present invention is even further characterized with ak_(off) rate to the p19 subunit of human IL-23 of from about 0.30×10⁻⁴sec⁻¹ to about 0.70×10⁻⁴ sec⁻¹. Preferably, the antibody of the presentinvention has a k_(off) rate to the p19 subunit of human IL-23 of about0.52×10⁻⁴ sec⁻¹.

The antibody of the present invention binds to the p19 subunit of humanIL-23 with high affinity. For the purposes of the present disclosure,the term “high affinity” refers to a K_(D) of at least about 21 pM. TheK_(D) values are established by binding kinetics at 37° C. as describedin the section entitled “Affinity Binding Measurement by Surface PlasmonResonance (BIAcore for Antibody I” in Example 1.

Unlike certain prior art antibodies that bind to human IL-23, theantibody of the present invention does not observably exhibit tissuecross-reactivity. In particular, the antibody of the present inventiondoes not observably bind to retinal tissue.

The antibody of the present invention possesses pharmaceuticallyacceptable physical-chemical properties, including pharmaceuticallyacceptable solubility in physiological and laboratory conditions, andpharmaceutically acceptable chemical and physical stability wherein theantibody remains in a monomeric form and very little high molecularweight (HMW) aggregates are observed under a range of conditions asdescribed in the section entitled “Physical-Chemical Properties of IL-23Antibody” in Example 1.

The present invention further provides pharmaceutical compositionscomprising an antibody of the present invention and one or morepharmaceutically acceptable carriers, diluents or excipients. Moreparticularly, the pharmaceutical compositions of the present inventionfurther comprise one or more additional therapeutic agents.

The present invention also provides a method of treating or preventing acondition in a patient, comprising administering to a patient in needthereof an effective amount of an antibody of the present invention,wherein the condition is an autoimmune or inflammatory conditionselected from the group consisting of multiple sclerosis, rheumatoidarthritis, psoriasis, inflammatory bowel diseases, ankylosingspondylitis, graft-versus-host disease, lupus and metabolic syndrome.

The present invention also provides a method of treating or preventing acondition in a patient, comprising administering to a patient in needthereof an effective amount of an antibody of the present invention,wherein the condition is cancer.

In an embodiment of the present invention, the cancer is melanoma,colon, ovarian, head and neck, lung, breast, or stomach cancer.

The present invention also provides the antibody of the presentinvention for use in therapy.

More particularly, the present invention provides the antibody of thepresent invention for use in the treatment or prevention of anautoimmune or inflammatory condition selected from the group consistingof multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatorybowel diseases, ankylosing spondylitis, graft-versus-host disease, lupusand metabolic syndrome.

The present invention also provides the antibody of the presentinvention for use in the treatment or prevention of cancer.

In an embodiment of the present invention, the cancer is melanoma,colon, ovarian, head and neck, lung, breast, or stomach cancer. Thepresent invention provides the use of an antibody of the presentinvention in the manufacture of a medicament for the treatment orprevention of a condition selected from the group consisting of multiplesclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases,ankylosing spondylitis, graft-versus-host disease, lupus and metabolicsyndrome.

The present invention also provides the use of an antibody of thepresent invention in the manufacture of a medicament for the treatmentor prevention of cancer.

In an embodiment of the present invention, the cancer is melanoma,colon, ovarian, head and neck, lung, breast, or stomach cancer.

The present invention also relates to polynucleotides encoding theabove-described antibody of the present invention.

The present invention provides a DNA molecule comprising apolynucleotide sequence encoding a light chain polypeptide having theamino acid sequence SEQ ID NO: 10.

The present invention also provides a DNA molecule comprising apolynucleotide sequence encoding a heavy chain polypeptide having theamino acid sequence SEQ ID NO: 9.

In one embodiment, the present invention provides a polynucleotideencoding an antibody of the present invention, wherein the HCVR isencoded by SEQ ID NO: 11 and the LCVR is encoded by SEQ ID NO: 12.

In a further embodiment, the present invention provides a polynucleotideencoding an antibody of the present invention, wherein the heavy chainis encoded by SEQ ID NO: 13 and the light chain is encoded by SEQ ID NO:14.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes cDNA, and synthetic DNA. TheDNA may be double-stranded or single-stranded. The coding sequences thatencode the antibody of the present invention may vary as a result of theredundancy or degeneracy of the genetic code.

The polynucleotides that encode for the antibody of the presentinvention may include the following: only the coding sequence for theantibody, the coding sequence for the antibody and an additional codingsequence such as a leader or secretory sequence or a pro-proteinsequence; the coding sequence for the antibody and non-coding sequence,such as introns or non-coding sequence 5′ and/or 3′ of the codingsequence for the protein. Thus the term “polynucleotide encoding anantibody” encompasses a polynucleotide that may include not only codingsequence for the protein but also a polynucleotide that includesadditional coding and/or non-coding sequence.

The polynucleotides of the present invention will be expressed in a hostcell after the sequences have been operably linked to an expressioncontrol sequence. The expression vectors are typically replicable in thehost organisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors will contain selectionmarkers, e.g., tetracycline, neomycin, and dihydrofolate reductase, topermit detection of those cells transformed with the desired DNAsequences.

The present invention provides a recombinant host cell comprising theDNA molecule of comprising a polynucleotide sequence encoding a lightchain polypeptide having the amino acid sequence SEQ ID NO: 10 and theDNA molecule comprising a polynucleotide sequence encoding a heavy chainpolypeptide having the amino acid sequence SEQ ID NO: 9, which cell iscapable of expressing an antibody comprising a heavy chain and a lightchain, wherein the amino acid sequence of the heavy chain is SEQ ID NO:9 and the amino acid sequence of the light chain is SEQ ID NO: 10.

The antibody of the present invention may readily be produced inmammalian cells such as CHO, NS0, HEK293 or COS cells; in bacterialcells such as E. coli, Bacillus subtilis, or Pseudomonas fluorescence;or in fungal or yeast cells. The host cells are cultured usingtechniques well known in the art.

The vectors containing the polynucleotide sequences of interest (e.g.,the polynucleotides encoding the polypeptides of the antibody andexpression control sequences) can be transferred into the host cell bywell-known methods, which vary depending on the type of cellular host.For example, calcium chloride transformation is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts.

Various methods of protein purification may be employed and such methodsare known in the art and described, for example, in Deutscher, Methodsin Enzymology 182: 83-89 (1990) and Scopes, Protein Purification:Principles and Practice, 3rd Edition, Springer, NY (1994).

The present invention provides a process for producing an antibody thatbinds to the p19 subunit of human IL-23 comprising a heavy chain and alight chain, wherein the heavy chain comprises amino acid sequence SEQID NO: 9 and light chain comprise amino acid sequences SEQ ID NO: 10,said process comprising the steps of:

-   -   a) cultivating a recombinant host cell of claim 7 under        conditions such that said antibody is expressed; and    -   b) recovering from said host cell the expressed antibody.

Further, the present invention provides a process for producing anantibody that binds to the p19 subunit of human IL-23 having a heavychain and a light chain, wherein the amino acid sequence of the heavychain is SEQ ID NO: 9 and the amino acid sequence of the light chain isSEQ ID NO: 10, said process comprising the steps of:

-   -   a) cultivating a recombinant host cell comprising a first        polynucleotide sequence encoding the polypeptide sequence given        by SEQ ID NO: 9 and a second polynucleotide sequence encoding        the polypeptide sequence given by SEQ ID NO: 10, under        conditions such that said polypeptide sequences are expressed;        and    -   b) recovering from said host cell an antibody comprising a heavy        chain and a light chain, wherein the polypeptide sequence of        said heavy chain is given by SEQ ID NO: 9 and the polypeptide        sequence of said light chain is given by SEQ ID NO: 10.

In one embodiment of the above-described process, the firstpolynucleotide sequence encoding the polypeptide sequence given by SEQID NO: 9 and the second polynucleotide sequence encoding the polypeptidesequence given by SEQ ID NO: 10 are part of the same nucleic acidmolecule.

In an embodiment, the present invention provides an antibody produced bythe afore-mentioned process.

In a further embodiment, the antibody produced by the afore-mentionedprocess has two heavy chains and two light chains, wherein thepolypeptide sequence of each heavy chain is given by SEQ ID NO: 9 andthe polypeptide sequence of each light chain is given by SEQ ID NO: 10.

The antibody of the present invention is an IgG type antibody and hasfour amino acid chains (two “heavy” chains and two “light” chains) thatare cross-linked via intra- and inter-chain disulfide bonds. Whenexpressed in certain biological systems, antibodies having native humanFe sequences are glycosylated in the Fc region. Antibodies may beglycosylated at other positions as well.

Each heavy chain is comprised of an N-terminal HCVR and a heavy chainconstant region (“HCCR”). Human heavy chains are classified as gamma,mu, alpha, delta, or epsilon, and define the isotype of an antibody asIgG, IgM, IgA, IgD, or IgE, respectively. Human IgG antibodies can befurther divided into subclasses, e.g., IgG₁, IgG₂, IgG₃, IgG₄.

Preferably, the antibody of the present invention contains an Fc portionwhich is derived from human IgG₄ Fc region because of a reduced abilityto engage Fc receptor-mediated inflammatory mechanisms or to activatecomplement resulting in reduced effector function.

More preferably, the antibody of the present invention contains anIgG₄-PAA Fc portion. The IgG₄-PAA Fe portion has a serine to prolinemutation at position 223 (S223P; SEQ ID NO: 9), a phenylalanine toalanine mutation at position 229 (F229A; SEQ ID NO: 9) and a leucine toalanine mutation at position 230 (L230A; SEQ ID NO: 9). The S223Pmutation is a hinge mutation that prevents half-antibody formation(phenomenon of dynamic exchange of half-molecules in IgG₄ antibodies).The F229A and L230A mutations further reduce effector function of thealready low human IgG₄ isotype.

Each heavy chain type is also characterized by a particular constantregion with a sequence well known in the art. The heavy chain constantregion is comprised of three domains (CH1, CH2, and CH3) for IgG.

Light chains are classified as kappa or lambda, which are eachcharacterized by a particular constant region as known in the art. Eachlight chain is comprised of a LCVR and a light chain constant region(“LCCR”). Preferably, the antibody of the present invention comprises akappa light chain.

The variable regions of each light/heavy chain pair form the antibodybinding site. The HCVR and LCVR regions can be further subdivided intoregions of hyper-variability, termed complementarity determining regions(“CDRs”), interspersed with regions that are more conserved, termedframework regions (“FR”). Each HCVR and LCVR is composed of three CDRsand four FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the threeCDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3” andthe three CDRs of the light chain are referred to as “LCDR1, LCDR2 andLCDR3”. The CDRs contain most of the residues which form specificinteractions with the antigen. There are currently three systems of CDRassignments for antibodies that are used for sequence delineation. TheKabat CDR definition (Kabat et al., “Sequences of Proteins ofImmunological Interest,” National Institutes of Health, Bethesda, Md.(1991)) is based upon antibody sequence variability. The Chothia CDRdefinition (Chothia et al., “Canonical structures for the hypervariableregions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917(1987); Al-Lazikani et al., “Standard conformations for the canonicalstructures of immunoglobulins”, Journal of Molecular Biology, 273,927-948 (1997)) is based on three-dimensional structures of antibodiesand topologies of the CDR loops. The Chothia CDR definitions areidentical to the Kabat CDR definitions with the exception of HCDR1 andHCDR2. The North CDR definition (North et al., “A New Clustering ofAntibody CDR Loop Conformations”, Journal of Molecular Biology, 406,228-256 (2011)) is based on affinity propagation clustering with a largenumber of crystal structures.

For the purposes of the present invention, a consensus of the threemethods is used to define CDRs. In the case of the light chain CDRs, theKabat and Chothia CDR definitions are used. In the case of HCDR1, ahybrid of the Kabat and Chothia CDR definitions is used. The Kabatdefinition of HCDR1 starts eight residues after the first cysteine ofthe heavy chain and is five residues in length, whereas the Chothiadefinition of HCDR1 starts three residues after this cysteine and isseven residues in length. The HCDR1 of the antibody of the presentinvention is defined by the Chothia starting position and the Kabat endposition. In the case of HCDR2, the Kabat CDR definition is used. In thecase of HCDR3, a hybrid of the North, Kabat and Chothia CDR definitionsis used. The Kabat definition of HCDR3 comprises residues 95-102 of theheavy chain (SEQ ID NO: 13 for the antibody of the present invention)and typically starts three residues after a cysteine. The Chothiadefinition of HCDR3 is the same as the Kabat definition. The Northdefinition of HCDR3 comprises residues 93-102 of the heavy chain (SEQ IDNO: 13 for the antibody of the present invention) and typically startsimmediately after the cysteine residue. The HCDR3 of the antibody of thepresent invention is defined by the North starting position and theKabat/Chothia/North end position.

Table 1 shows exemplary CDR assignments of the antibody of the presentinvention.

TABLE 1 CDR Assignments CDR Start Definition End Definition LCDR1Kabat/Chothia/North Kabat/Chothia/North LCDR2 Kabat/ChothiaKabat/Chothia/North LCDR3 Kabat/Chothia/North Kabat/Chothia/North HCDR1Chothia Kabat/North HCDR2 Kabat/North Kabat HCDR3 NorthKabat/Chothia/North

An antibody of the present invention is an engineered antibody that hasbeen designed to have frameworks, hinge regions, and constant regions ofhuman origin that are identical with or substantially identical(substantially human) with frameworks and constant regions derived fromhuman genomic sequences. Fully human frameworks, hinge regions, andconstant regions are those human germline sequences as well as sequenceswith naturally-occurring somatic mutations and those with engineeredmutations. An antibody of the present invention may comprise framework,hinge, or constant regions derived from a fully human framework, hinge,or constant region containing one or more amino acid substitutions,deletions, or additions therein. Further, an antibody of the presentinvention is preferably substantially non-immunogenic in humans.

A variety of different human framework sequences may be used singly orin combination as a basis for an antibody of the present invention.Preferably, the framework regions of an antibody of the presentinvention are of human origin or substantially human (at least 95%, 97%or 99% of human origin.) The sequences of framework regions of humanorigin may be obtained from The Immumunoglobulin Factsbook, byMarie-Paule Lafranc, Gerard Lefranc, Academic Press 2001, ISBN012441351.

The framework sequence for an antibody of the present invention servesas the “donor” variable framework region and can be used to createadditional antibodies with the same CDRs specified herein usingmethodology known in the art. Furthermore, the framework sequence for anantibody of the present invention can be compared to other known humanframework sequences to generate additional antibodies. Thus, thisinformation can be used to “back-mutate” another selected homologoushuman framework region to the donor amino acid residue at thesepositions. Further, any “rare” amino acids can be detected in additionalhuman frameworks such that the consensus or donor amino acid residue canbe used at the relevant position.

Methods for producing and purifying antibodies are well known in the artand can be found, for example, in Harlow and Lane (1988) Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory Press. Cold SpringHarbor, N.Y., Chapters 5-8 and 15. For example, mice can be immunizedwith human IL-23, or fragments thereof, and the resulting antibodies canthen be recovered, purified and the amino acid sequences determinedusing conventional methods well known in the art. The antibody of thepresent invention is engineered to contain one or more human frameworkregions surrounding CDRs derived from a non-human antibody. Humanframework germline sequences can be obtained from ImMunoGeneTics (IMGT)via their website http://imgt.cines.fr, or from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001,ISBN 012441351. Particular, germline light chain frameworks for use inthe antibody of the present invention include 02.

Particular germline heavy chain framework regions for use in theantibody of the present invention include VH1-69.

The engineered antibodies of the present invention may be prepared andpurified using known methods. For example, cDNA sequences encoding aheavy chain (for example, the amino acid sequence given by SEQ ID NO: 9)and a light chain (for example, the amino acid sequence given by SEQ IDNO: 10) may be cloned and engineered into a GS (glutamine synthetase)expression vector. The engineered immunoglobulin expression vector maythen be stably transfected in CHO cells. Mammalian expression ofantibodies will result in glycosylation, typically at highly conservedN-glycosylation sites in the Fc region. Stable clones may be verifiedfor expression of an antibody specifically binding to human IL-23.Positive clones may be expanded into serum-free culture medium forantibody production in bioreactors. Media, into which an antibody hasbeen secreted, may be purified by conventional techniques. For example,the medium may be conveniently applied to a Protein A or G Sepharose FFcolumn that has been equilibrated with a compatible buffer, such asphosphate buffered saline. The column is washed to remove nonspecificbinding components. The bound antibody is eluted, for example, by pHgradient and antibody fractions are detected, such as by SDS-PAGE, andthen pooled. The antibody may be concentrated and/or sterile filteredusing common techniques. Soluble aggregate and multimers may beeffectively removed by common techniques, including size exclusion,hydrophobic interaction, ion exchange, or hydroxyapatite chromatography.The product may be immediately frozen, for example at −70° C., or may belyophilized.

The antibodies of the present invention are monoclonal antibodies.“Monoclonal antibody” or “mAb”, as used herein, refers to an antibodythat is derived from a single copy or clone including, for example, anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced. Monoclonal antibodies thereof can be produced, for example,by hybridoma technologies, recombinant technologies, phage displaytechnologies, synthetic technologies, e.g., CDR-grafting, orcombinations of such or other technologies known in the art.

In another embodiment of the present invention, the antibody, or thenucleic acid encoding the same, is provided in isolated form.

The antibody of the present invention, or pharmaceutical compositionscomprising the same, may be administered by parenteral routes (e.g.,subcutaneous, intravenous, intraperitoneal, intramuscular, ortransdermal).

Pharmaceutical compositions of the present invention can be prepared bymethods well known in the art (e.g., Remington: The Science and Practicea/Pharmacy, 19^(th) edition (1995), (A. Gennaro et al., Mack PublishingCo.) and comprise an antibody as disclosed herein, and one or morepharmaceutically acceptable carriers, diluents, or excipients. Forexample, an antibody of the present invention can be formulated withagents such as sodium citrate, citric acid, polysorbate 80, sodiumchloride and sucrose and the resulting composition may then belyophilized and stored at 2° C.-8° C. The lyophilized composition maythen be reconstituted with sterile water for injection prior toadministration.

The term “bind (or “binds”) to the p19 subunit of human IL-23”, as usedherein, refers to a detectable interaction of the antibody of thepresent invention with an epitope on the p19 subunit of human IL-23given by the amino acid sequence of SEQ ID NO: 15. The interactionbetween the antibody of the present invention and the p19 subunit ofhuman IL-23 is measured by binding kinetics at 37° C. as described inthe section entitled “Affinity Binding Measurement by Surface PlasmonResonance (BIAcore) for Antibody I” in Example 1.

The term “epitope” as used herein refers to amino acid residues that lieclose together on the protein (antigen) surface and interact with anantibody. There are two broad classes of epitopes: linear epitopes andconformational epitopes.

The term “linear epitope” as used herein refers to a continuous primaryamino acid sequence of a particular region of a protein.

The term “conformational epitope” as used herein refers to discontinuoussections of the antigen's amino acid sequence that are contacted by theantibody of the invention. Conformational epitopes are defined by thestructure as well as the sequence of the native protein; these epitopesmay be continuous or discontinuous. Components of the epitope can besituated on disparate parts of the protein, which are brought close toeach other in the folded native protein structure. In the context of thepresent invention, the antibody of the present invention binds to aconformational epitope within amino acid positions 81-99 and 115-140 ofSEQ ID NO: 15, wherein the antibody contacts at least amino acidsresidues 94P, 95S, 97L, 98P, 99D, 123W, 130S, 133P and 137W of SEQ IDNO: 15. The conformational epitope is not, however, limited to theseamino acid residues and may comprise additional amino acid residueswithin amino acid positions 81-99 and 115-140 of SEQ ID NO: 15.

The term “does not observably bind retinal tissue”, as used herein,refers to the absence of a detectable interaction of the antibody of thepresent invention with human and cynomolgus monkey retinal tissue. Theinteraction between the antibody of the present invention and the humanand cynomolgus monkey retinal tissue is assessed in animmunohistochemistry assay as described in the section entitled “RetinalTissue Cross-Reactivity: In Vitro Analysis by Immunohistochemistry” inExample 1. The term “observably” as used in the present context refersto a visual assessment of the human and cynomolgus monkey retinal tissueto determine if the antibody of the present invention binds to saidhuman and cynomolgus monkey retinal tissue.

The term “selective” as used herein in reference to an antibody of thepresent invention refers to an antibody that binds the p19 subunit ofhuman IL-23 but does not bind to the p40 subunit shared by human IL-23and human IL-12.

The term “neutralizing” refers to “neutralizing antibody”, as usedherein, is intended to refer to inhibition of the biological activity ofhuman IL-23. Measuring one or more indicators of IL-23 biologicalactivity as determined using either the mouse splenocyte bioassay (seesection entitled “In Vitro Neutralization of Human or Cynomolgus MonkeyIL-23 by Antibody I in Murine Splenocytes in Example 1) or the humanIL-23 neutralization assay (see section entitled “Neutralization ofHuman IL-23: Acute, Local” in Example 1) can assess this inhibition ofthe biological activity of human IL-23.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction. Itis calculated by the formula:

K _(off) /K _(on) =K _(D)

The term “k_(on)”, as used herein, is intended to refer to theassociation or on rate constant, or specific reaction rate, of theforward, or complex-forming, reaction, measured in units: M⁻¹sec⁻¹.

The term “k_(off)”, as used herein, is intended to refer to thedissociation or off rate constant, or specific reaction rate, fordissociation of an antibody from the antibody/antigen complex, measuredin units: sec⁻¹.

The term “IC₅₀”, as used herein, is intended to refer to the effectiveconcentration of antibody of the present invention needed to neutralize50% of the bioactivity of IL-23 on mouse splenocytes in the bioassaydescribed in the section entitled “In Vitro Neutralization of Human orCynomolgus Monkey IL-23 by Antibody I in Murine Splenocytes in Example1.

The term “polynucleotide”, as used herein, is intended to include DNAmolecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded.

The term “isolated”, as used herein, refers to a protein, peptide ornucleic acid which is free or substantially free from othermacromolecular species found in a cellular environment.

The term “substantially free”, as used herein, means the protein,peptide or nucleic acid of interest comprises more than 80% (on a molarbasis) of the macromolecular species present, preferably more than 90%and more preferably more than 95%.

A “patient” is a mammal, preferably a human.

The term “treating” (or “treat” or “treatment”) refers to slowing,interrupting, arresting, alleviating, stopping, reducing, or reversingthe progression or severity of an existing symptom, disorder, condition,or disease

The term “effective amount”, as used herein, refers to the amount ordose of an antibody of the present invention which, upon single ormultiple dose administration to the patient, provides the desired effectin the patient under treatment. An effective amount can be readilydetermined by the attending diagnostician, as one skilled in the art. byconsidering a number of factors such as the species of mammal; its size,age, and general health; the specific disease involved; the degree orseverity of the disease; the response of the individual patient; theparticular antibody administered; the mode of administration; thebioavailability characteristics of the preparation administered; thedose regimen selected; and the use of any concomitant medications.

EXAMPLE

The following Example further illustrates the invention. It isunderstood, however, that the Example is set forth by way ofillustration and not limitation, and that various modifications may bemade by one of ordinary skill in the art.

Example 1 Production of Antibodies

Antibody I of this example comprises two heavy chains and two lightchains, each heavy chain having the amino acid sequence given by SEQ IDNO: 9 and each light chain having the amino acid sequence given by SEQID NO: 10. Antibody I can be made and purified as follows. Anappropriate host cell, such as HEK 293 or CHO, is either transiently orstably transfected with an expression system for secreting antibodiesusing an optimal predetermined HC:LC vector ratio or a single vectorsystem encoding both heavy chain (SEQ ID NO: 9) and light chain (SEQ IDNO: 10). Clarified media, into which the antibody has been secreted, ispurified using any of many commonly-used techniques. For example, themedium may be conveniently applied to a Protein A or G column that hasbeen equilibrated with a compatible buffer, such as phosphate bufferedsaline (pH 7.4). The column is washed to remove nonspecific bindingcomponents. The bound antibody is eluted, for example, by pH gradient(such as 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium citratebuffer pH 2.5). Antibody fractions are neutralized (for example byadding 1/10^(th) volume of 1M TRIS at pH 8.0), detected, such as bySDS-PAGE, and then are pooled. Further purification is optional,depending on the intended use. The antibody may be concentrated and/orsterile filtered using common techniques. Soluble aggregate andmultimers may be effectively removed by common techniques, includingsize exclusion, hydrophobic interaction, ion exchange, or hydroxyapatitechromatography. The purity of the antibody after these chromatographysteps is greater than 99%. The product may be immediately frozen at −70°C. or may be lyophilized.

Affinity Binding Measurement by Surface Plasmon Resonance (BIAcore)

Antibody affinity (K_(D)) to human, cynomolgus monkey or rabbit IL-23 isdetermined using a BIAcore Biosensor 2000 and BIAevaluation softwarewith a 1:1 binding with mass transfer model. A capture protein (ProteinA, Calbiochem) is coupled via free amine groups to carboxyl groups onflow cells 1 and 2 of a CM4 biosensor chip using a mixture ofN-ethyl-N-(dimethylaminopropyl)-carbodiimide (EDC) andN-hydroxysuccinimide (NHS). Flow cells are monitored with a flow rate of80 μL/minute using a buffer containing 0.01 M HEPES, pH 7.4, 150 mMNaCl, 0.005% surfactant P20. Antibody I is captured on flow cell 2 toyield a total of 40 to 60 response units (RU). Multiple cycles ofincreasing concentrations of IL-23 are then injected over flow cells 1and 2 (0.62 nM to 30 nM for human and monkey IL-23 and 30 nM to 240 nMfor rabbit IL-23) followed by a regeneration step using glycine-HCl (pH1.5) between each cycle. Flow cell 1 is used as a control to monitornon-specific binding of IL-23 and the data reflects flow cell 2 minusflow cell 1. Each cycle includes an antibody capture step followed byinjection of IL-23 at one concentration with a 30 minute dissociationperiod, then regeneration. Two cycles where buffer is injected in placeof IL-23, serve as a control for baseline subtraction and correct fordrift associated with the dissociation of Antibody I from the protein Asurface. Affinity is measured at 37 ° C. The assay is performed 2 timeswith human, monkey or rabbit IL-23. Antibody I is tested 2 times eachwith mouse IL-23 at 333 nM, rat IL-23 at 200 nM, human IL-12 at 333 nM,human IL-27 at 500 nM or human IL-35 at 833 nM.

The on-rate (k_(on)) and off-rate (k_(off)) for each antigen areevaluated using a 1:1 binding with mass transfer model. The affinity(K_(D)) is calculated from the binding kinetics according to therelationship: K_(D)=k_(off)/k_(on).

TABLE 2 Binding Parameters for Antibody I On Rate (k_(on)) Off Rate(k_(off)) Affinity (K_(D) ^(a)) (Avg ± SD) (Avg ± SD) (Avg ± SD) Antigen(M⁻¹s⁻¹) (10⁶) (s⁻¹) (10⁻⁴) (PM) Human No detectable No detectable Nodetectable IL-12 binding binding binding Human 2.43 ± 0.16 0.52 ± 0.21   21 ± 9.9 IL-23 Human No detectable No detectable No detectable IL-27binding binding binding Human No detectable No detectable No detectableIL-35 binding binding binding Monkey 1.28 ± 0.05  0.7 ± 0.11    55 ± 6.4IL-23 Rabbit 0.09 ± 0.001 47.9 ± 0.4 53,000 ± 1131 IL-23 Mouse Nodetectable No detectable No detectable IL-23 binding binding binding RatNo detectable No detectable No detectable IL-23 binding binding binding^(a)Calculated as K_(D) = k_(off)/k_(on) n = 2 for each antigen. IL-12was tested at a 400x concentration of what is detectable for IL-23.IL-27 and IL-35 were tested at an 800x concentration of what isdetectable for IL-23. Mouse and rat IL-23 were tested at 500x and 300xconcentrations of what is detectable for human IL-23.

Antibody I produces a concentration-dependent binding response withhuman, cynomolgus monkey, and rabbit IL-23 using this method. Saturationof binding of IL-23 is attained at a concentration of 30 nM (human andmonkey) and 240 nM (rabbit) using 80-100 response units of Antibody Icaptured on the chip surface. Under the conditions tested, the bindingaffinity (K_(D)) of human, monkey, or rabbit IL-23 to Antibody I is 21,55 or 53,000 pM respectively (Table 1). Mouse IL-23, rat IL-23, humanIL-12, human IL-27 or human IL-35 do not bind to Antibody I under theseconditions.

In Vitro Inhibition of IL-23 Binding to IL-23 Receptor

Recombinant human IL-23R/Fc is coupled via free amine groups to carboxylgroups on flow cell 2 of a CM4 biosensor chip using a mixture ofN-ethyl-N-(dimethylaminopropyl)-carbodiimide (EDC) andN-hydroxysuccinimide (NHS). Recombinant human IgG₁ Fc (R&D Systems,Inc.) is coupled using the same method to flow cell 1 of the same chip.Mouse anti-6X HIS antibody (R&D Systems, Inc.) is coupled using the samemethod to flow cell 4 of the same chip. Mouse anti-6X HIS is used topre-capture human IL-12Rβ1/Fc (R&D Systems, Inc.) which contains a HIStag. Flow cells are monitored with a flow rate of 30 μL/minute using abuffer containing 0.01 M HEPES, pH 7.4, 150 mM NaCl, 0.005% surfactantP20. Recombinant human IL-23 is pre-incubated for 90 minutes with orwithout the addition of a 16× molar excess of Antibody I. Eachcombination is injected over flow cells 1, 2 and 4 in a total volume of150 μL followed by a regeneration step using glycine-HCl (pH 1.5)between each test. Flow cell 1 is used as a control to monitornon-specific binding of IL-23 to the chip. BIAevaluation software isused to prepare overlays of individual binding sensorgrams.

Antibody I neutralizes human IL-23 using in vitro functional assays.Furthermore, Antibody I prevents binding of IL-23 to IL-23R/Fc. The datain Table 3 shows:

-   -   (A) IL-23 binds to IL-23R/Fc;    -   (B) Antibody I/IL-23 complex does not bind to IL-23R/Fc;    -   (C) IL-23 binds to IL-12Rβ1/Fc, and    -   (D) Antibody I/IL-23 complex binds to IL-12Rβ1/Fc.

TABLE 3 Effect of Antibody I on IL-23 binding to IL-23R CytokineAntibody Binding to IL-23R Binding to IL-12β1 IL-23 None YES YES IL-23 INO YES

Thus, Antibody I neutralizes IL-23 because it inhibits the binding ofIL-23 to the IL-23R subunit. Additionally, Antibody I does not inhibitbinding of IL-23 to the IL-12Rβ1 subunit.

In Vitro Neutralization of Human or Cynomolgus Monkey IL-23 by AntibodyI in Murine Splenocytes

For evaluation of Antibody I, a concentration of human or cynomolgusmonkey IL-23 that gives approximately 50% of maximal production of IL-17is used (16 pM). A dose response ranging from 800,000 to 4.4 pM ofAntibody I is evaluated. Antibody I or an IgG₄ control antibody iscombined with human or cynomolgus monkey IL-23 in a separate well for 90minutes at 37° C. before addition to the cells (pre-incubation mix).

Splenocytes from C57BL/6 mice stimulated with IL-23 and IL-2 produceIL-17 (Aggarwal, S. et al., “Interleukin-23 Promotes a Distinct CD4 TCell Activation State Characterized by the Production ofInterleukin-17”, Journal of Biological Chemistry, 278 (3): 1910-19142003). Mouse splenocytes are re-suspended at 5×10⁶ WBC/mL in assay media(RPMI1640 with L-glutamine containing 10% FBS, 1% non-essential aminoacids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mlstreptomycin, 0.00035% 2-mercaptoethanol, 50 ng/mL human IL-2) anddispensed in volumes of 100 μL per well into a 96-well culture plate.The pre-incubation mix of Antibody I/IL-23 is dispensed as 100□ μL atper well and incubated at 37° C. in 5% CO₂. Forty-eight hours later,culture supernatants are tested for mIL-17 using a commercial ELISA kitfrom R&D Systems (DY421) according to the instructions in the kit usingduplicate wells at each dilution. An IC₅₀ is determined using a 4parameter curve fit of the data.

Mouse splenocytes produce IL-17 in response to human or cynomolgusmonkey IL-23. Antibody I neutralizes human or cynomolgus monkey IL-23.The calculated IC₅₀ is 82±11 pM for human and 120±14 pM for cynomolgusmonkey IL-23, n=2 for each (Table 4). These results demonstrate thatAntibody I is able to neutralize human or cynomolgus monkey IL-23 invitro.

TABLE 4 IC₅₀ in the in vitro human and cynomolgus monkey IL-23neutralization assay Species Assay # Antibody IC₅₀ (pM) Human 1 I 90Human 2 I 74 Human Average (SD)  82 (11) Cyno 3 I 110 Cyno 4 I 130 CynoAverage (SD) 120 (14)

Neutralization of Human IL-23: Acute, Local

Animals (C57BL/six females, eight weeks old from Jackson Labs) arehoused (minimum of 72 hrs after arrival) and fed normally prior to theexperiment and for the duration of the study. Hair is removed from theback of mice with electric clippers, and 3 days later mice (n=10 pergroup) received a subcutaneous injection of Antibody I or an IgG₄isotype control antibody (0.54 mg per mouse). The following 2 days, miceare injected intradermally with human IL-23 in one location on one sideof the back (1 μg in 50 μL diluted with sterile saline) using a 29-gaugeneedle. Sterile saline is used as a vehicle control on the other side ofthe back. Mice are sacrificed 24 hours after the last human IL-23injection and skin samples are removed from IL-23-injected side and fromthe sterile saline-injected side, keeping at least 5 mm away from thehair boundary. Skin samples are frozen directly in liquid nitrogen formRNA studies.

Total RNA is isolated from frozen skin tissue by homogenization inLysing Matrix A shaker tubes (Qbiogene Inc./Bio101 Systems) followed byRNeasy Mini kit cleanup (Qiagen, Inc.). RNA concentrations aredetermined from spectrophotometric absorption at 260 nm. RNA isreverse-transcribed into cDNA using High-Capacity cDNA ReverseTranscription Kit (PE Applied Biosystems). All reactions are performedin triplicate on an ABI Prism 7900HT (PE Applied Biosystems) todetermine the relative abundance of assayed mRNAs. Primer probe sets formouse IL-17A (Mm00439618_m1), mouse IL-17F (Mm00521423_m1) and mousekeratin-16 (Mm00492979_g1) are obtained from PE Applied Biosystems. Both18S and GAPD are measured as endogenous controls to normalizevariability in gene expression levels. Expression data is analyzed usingDelta (Δ-Δ) Ct method. Individual Ct values are calculated as means oftriplicate measurements. Experiments are performed two times. Unpairedt-test is used where appropriate. P<0.05 is considered to bestatistically significant.

To explore whether systemic administration of Antibody I is able toneutralize the local response to human IL-23, human IL-23 protein isinjected intradermally into mice to investigate the downstreamconsequences of cutaneous IL-23 exposure. Skin from wild-type micetreated saline solution daily does not show detectable levels of mouseIL-17A or mouse IL-17F.

However, injection of human IL-23 induces mRNA expression of mouseIL-17A and mouse IL-17F (Table 5). Treatment with Antibody I but notisotype control antibody abrogated the human IL-23-induced IL-17A andIL-17F mRNA expression.

TABLE 5 In vivo neutralization of human IL-23 induced murine IL-17A andIL-17F mRNA expression. Ct Values PBS IL-23 IL-17A IL-17F IL-17A IL-17FIsotype control ≧40 ≧40 35.4 31.6 Antibody I ≧40 ≧40 ≧40 ≧40

Furthermore, human IL-23 injection induces an epidermal thickeningassociated with increased expression of keratin-16, aproliferation-associated cytokeratin. The induction of keratin-16 issignificantly inhibited by administration of Antibody I (fold inductionof murine keratin-16 is 5.21±2.72 for isotype control antibody versus1.23±0.72 for Antibody I; p=0.0003).

All together, these results show that Antibody I effectively inhibitshuman IL-23-induced mouse IL-17A, IL-17F and keratin-16 mRNA productionin an acute local in vivo assay.

Retinal Tissue Cross-Reactivity: In Vitro Analysis byImmunohistochemistry

Sections of fresh-frozen human and cynomolgus monkey retinal tissue (5-7μm thick) are cut on a cryostat. The sections are fixed in acetone forapproximately 10 minutes at room temperature, allowed to dry overnightat room temperature and stored at approximately −80° C. until use.Acetone-fixed slides are subsequently removed from the freezer andallowed to dry overnight at room temperature. The following steps areperformed at room temperature. The slides are incubated in 1×Morphosave™ for approximately 15 minutes to preserve morphology. Theslides are washed 10 minutes in 1×PBS and then incubated in 0.3% H₂O₂ in1×PBS at room temperature for approximately 20 minutes to quenchendogenous peroxidase activity. After incubation, the slides are washedtwo times for approximately 5 minutes in 1×PBS. Endogenous biotin isblocked by sequential incubation (approximately 15 minutes each) inavidin and biotin solutions. Following the incubation in biotin, thetissue sections are blocked with a blocking antibody solution for 30minutes. Antibody I or control human IgG₄ is applied to sections at theoptimal concentrations (2.5 or 5 μg/mL) or five times the optimalconcentration (25 μg/mL) and incubated for 1 hour at room temperature.Slides are then rinsed and incubated with biotinylated mouse anti-humanIgG₄ antibody (2.5 mg/mL) for 30 minutes. Bound primary/secondaryantibody complexes are detected with streptavidin-biotin-horseradishperoxidase conjugate and a diaminobenzidine chromagen substrate.

CHO cells transfected with human IL-23 are used as a positive controlsample in all experiments. Parental CHO (non-transfected) cells are usedas a negative control sample and did not stain. Binding is not observedin serial sections stained with the isotype control antibody (humanIgG4). Antibody I does not observably bind retinal tissue.

Epitope Mapping for Antibody I: Alanine Scanning

Background to Epitope Mapping using Yeast Displayed Antigen

Epitope mapping studies are performed to determine the specific aminoacids in the human IL-23 p19 subunit (SEQ ID NO: 15) that are requiredfor Antibody I binding. Epitope mapping of Antibody I is completed byutilizing alanine scanning in conjunction with a yeast display platform.

Exposed amino acid positions of the p19 subunit of human IL-23 areidentified by analysis in PyMOL. The exposed or partially exposedpositions of the p19 subunit of IL-23 are shown in Table 6. Thosepositions that were determined not to be exposed are omitted from thisstudy, i.e. only amino acid positions of the p19 subunit of human IL-23that are exposed or partially exposed are mutated. Accordingly, not allpositions are investigated.

Although the epitope mapping is only performed on the p19 subunit ofIL-23 (no epitope mapping performed on the p40 subunit of IL-23 asAntibody I does not detectably bind to the p40 subunit), both the p19subunit and p40 subunit of human IL-23 must be co-expressed in the yeastdisplay platform.

Single yeast displayed alanine mutants of the p19 subunit of human IL-23are constructed and antibody binding determined in order to identify theepitope. By measuring the affinity of antibody mutants compared to thewild-type yeast displayed antigen, it is possible to determine theenergetic contribution of the amino acid side chain to antibody binding.

Mutant Library Construction

The p40 gene is cloned into the soluble-expression plasmid, pYKY, whichhas a uracil selection marker. The p19 subunit gene is cloned into theyeast display plasmid, pEMD3, which contains a tryptophan selectionmarker and a VS tag at the N-terminus and to a GPDL2 anchor protein atthe C-terminus allowing display on the surface of yeast under thetryptophan selectable marker. The restriction sites used for cloning areXhoI and BamHI in the pYKY plasmid and AvrII and XmaI in the pEMD3plasmid, respectively.

Alanine mutations are introduced at every exposed position and testedfor double positive staining with V5 antibody and Antibody I. Panels ofp19 alanine mutants are constructed in pEMD3 plasmids using sitedirected mutagenesis (Kunkel Mutagenesis). Briefly, uracil containingssDNA of the pEMD3 vector is produced after transformation into CJ236(New England Biolabs). A single colony of the transformation is grownovernight and the ssDNA rescued following infection with M13K07 helperphage (New England Biolabs) and ssDNA purified using a QIAprep spin M13kit. Oligonucleotides encoding alanine mutations are annealed at a 20:1molar ratio to the uracil template by denaturing at 85° C. for 5minutes, ramping to 55° C. over 1 hour, holding at 55° C. for 5 minutes,then chilling on ice. Second strand synthesis is then completed with T4polymerase, T4 ligase and dNTPs (Invitrogen). The reaction iselectroporated into Top10 E. coli (Invitrogen) and single coloniespicked, dsDNA prepared using the QIAprep miniprep kit (Qiagen) andmutations confirmed by sequencing. p19 mutants are then co-transformedinto BJ5464 yeast (ATCC) with the p40 pYKY plasmid and grown in completeminimal media without tryptophan and uracil.

Selection of Mutated Antigen Library for Loss of Antigen Binding

In order to identify the antibody epitope, the mutated antigen libraryis selected for loss of antibody binding by flow cytometry. Yeast cellsare stained with two antibodies, one of which is being mapped and one ofwhich is not. Yeast-displayed antigen mutants are selected for loss ofbinding to the first antibody, but retention of binding to the secondantibody. Retention of binding of the second antibody ensures thatmutants are selected on the basis of mutations in the epitope, ratherthan selection of unfolded or poorly displayed mutants.

For the present analysis, the first antibody (i.e. the antibody whoseepitope is being mapped) is Antibody I and the second antibody is ananti-V5 antibody. Yeast are stained with anti-V5 antibody (Invitrogen)and Antibody I to begin with and subsequently with a secondary goatanti-mouse IgG_(2a) (Invitrogen, Alexa Fluor® 647) to detect anti-V5antibody (expression/display) and a goat anti-human kappa RPE (SouthernBiotech) to detect Antibody I. Yeast are analyzed by flow cytometry on aBecton Dickinson LSRII, where 50,000 events are collected based ongating cells by light scatter, V5/Alexa647 and Antibody I/PE staining.Data analysis for binding of each of Antibody I and anti-V5 antibody isperformed using FACSDiva v6.1.2 software, which calculates thepercentage of double stained yeast cells.

Results

Due to displayed protein partition, at best 50% of yeast will displayIL-23 p19. Detection of double-positive yeast cells demonstrate that theamino acid position under investigation is not involved with Antibody Ibinding to IL-23. Detection of only V5 staining demonstrates that theprotein is expressed and displayed on the surface of the yeast and thatthe amino acid position under investigation is important for Antibody Ibinding. It is determined that those residues that demonstrated >50%reduction in double positive staining compared to adjacent residues areimportant for binding. These residues are highlighted in Table 7. Somepositions demonstrate the lack of both V5 and Antibody I binding,suggesting that amino acid residue may be necessary for proteinconformation. Systematic investigation of each exposed or partiallyexposed amino acid position in the IL-23 p19 subunit (SEQ ID NO: 15)demonstrates that positions 94P, 95S, 97L, 98P, 99D, 123W, 130S, 133P,and 137W are important for Antibody I binding to human IL-23 based onthe reduced amount of double positive staining for V5 and Antibody Ibinding (Table 7).

Epitope mapping is also performed using hydrogen-deuterium exchange. Theresults of this hydrogen-deuterium exchange epitope mapping illustratethat the epitope of Antibody I is a conformational epitope withinresidues 81-99 and 115-140 of human IL-23 (SEQ ID NO: 15).

TABLE 6 Exposed or partially exposed amino acid sequence of mature humanIL-23 p19 subunit Position 1 2 3 4 5 6 7 8 9 10 Amino Acid R A V P G G SS P A Exposed/Partially x x x x x x x x x exposed Position 11 12 13 1415 16 17 18 19 20 Amino Acid W T Q C Q Q L S Q K Exposed/Partially x x xx x x x exposed Position 21 22 23 24 25 26 27 28 29 30 Amino Acid L C TL A W S A H P Exposed/Partially x x x x x exposed Position 31 32 33 3435 36 37 38 39 40 Amino Acid L V G H M D L R E E Exposed/Partially x x xx x x x x x x exposed Position 41 42 43 44 45 46 47 48 49 50 Amino AcidG D E E T T N D V P Exposed/Partially x x x x x x x x x exposed Position51 52 53 54 55 56 57 58 59 60 Amino Acid H I Q C G D G C D PExposed/Partially x x x x x exposed Position 61 62 63 64 65 66 67 68 6970 Amino Acid Q G L R D N S Q F C Exposed/Partially x x x x x x xexposed Position 71 72 73 74 75 76 77 78 79 80 Amino Acid L Q R I H Q GL I F Exposed/Partially x x x x x exposed Position 81 82 83 84 85 86 8788 89 90 Amino Acid Y E K L L G S D I F Exposed/Partially x x x x x xexposed Position 91 92 93 94 95 96 97 98 99 100 Amino Acid T G E P S L LP D S Exposed/Partially x x x x x x x x x x exposed Position 101 102 103104 105 106 107 108 109 110 Amino Acid P V G Q L H A S L LExposed/Partially x x x x x x exposed Position 111 112 113 114 115 116117 118 119 120 Amino Acid G L S Q L L Q P E G Exposed/Partially x x x xx x exposed Position 121 122 123 124 125 126 127 128 129 130 Amino AcidH H W E T Q Q I P S Exposed/Partially x x x x x x x x x exposed Position131 132 133 134 135 136 137 138 139 140 Amino Acid L S P S Q P W Q R LExposed/Partially x x x x x x x x x x exposed Position 141 142 143 144145 146 147 148 149 150 Amino Acid L L R F K I L R S L Exposed/Partiallyx x x x x x exposed Position 151 152 153 154 155 156 157 158 159 160Amino Acid Q A F V A V A A R V Exposed/Partially x x x x exposedPosition 161 162 163 164 165 166 167 168 169 170 Amino Acid F A H G A AT L S P Exposed/Partially x x x x x x x x x x exposed

Physical-Chemical Properties of IL-23 Antibody

Antibody I has pharmaceutically acceptable solubility, chemicalstability and physical stability.

A. Solubility

Sufficiently high solubility is desired to enable convenient dosing. Forexample, a 1 mg/kg dose administered by a 1.0 mL injection into a 100 kgpatient will require solubility of 100 mg/mL. In addition, maintainingthe antibody in a monomeric state without high molecular weight (HMW)aggregation at high concentration is also desirable.

Antibody I is formulated at approximately 1 mg/mL in aphysiological-like buffer (PBS, pH 7.4) and under two drug productformulation conditions (10 mM citrate, pH 6, plus and minus 150 mMNaCl). The antibody is centrifuged at 2000×G through an Amicon Ultra 30kDa molecular weight filter (Millipore, UFC803204) to concentrate theantibody while maintaining the same buffer conditions. Centrifugation iscontinued until solubility limit or minimal holdup volume of the deviceis reached. Greater than 100 mg/mL solubility is achieved under allthree conditions.

Size-exclusion chromatography (SEC) is used to assess whether anincrease in high molecular weight (HMW) polymer occurred followingconcentration of the antibody formulations to greater than 1.00 mg/mL.The starting antibody solution and concentrated antibody solution areinjected onto a TSK3000 SWXL column (TOSOH Bioscience) using a mobilephase consisting of 12 mM phosphate, 500 mM NaCl, pH 7.4. No largeincrease in soluble polymer is observed under any formulation conditiontested (<0.6% HMW polymer by SEC).

B. Chemical Stability

Antibody I is formulated at 1 mg/mL in 10 mM buffer (10 mM citrate forpH 4, 5, 6, and 7; 10 mM TRIS for pH 8) and incubated for 4 weeks at 4,25, or 40° C. Chemical stability is monitored by SEC (see above method),cation exchange chromatography [CEX; Dionex, using a gradient betweenBuffer A (20 mM sodium phosphate, pH 5.8, 0.36% CHAPS) and Buffer B (20mM sodium phosphate, pH 5.8, 0.36% CHAPS, 200 mM sodium chloride)],CE-SDS (Agilent Bioanalyzer with a protein 230 chip under reducingconditions) and by LC-MS characterization of enzymatically digestedmaterial.

Antibody I is stable against polymer formation (SEC) over pH 5-8 evenafter 4-weeks at 40° C. At pH 4, significant polymer is observed at 40°C. but not at 25° C. (4 wk). Expected peptide bond hydrolysis orclipping is evident at pH 4 (40° C.) by CE-SDS. The degradation level atpH 4.0 is typical of IgG₄ antibodies. Above pH 4 (pH 5-8) levels are lowand do not consistently change with time and thus likely representbackground noise.

This hypothesis is consistent with the LC-MS analysis which detects noclipping at pH 6 while measuring typical level of antibody clipping atpH 4. Changes in charged variants are monitored by CEX. The startingmaterial consists of three significant main peaks which minimizeresolving power of this assay. In general, the 25° C. and 40° C.stressed samples are higher than the 4° C. control, but levels did notincrease with incubation time (actually decreased in many cases). Thepercent change at pH 6.0 (4 wk at 25° C. minus 4° C. control) is 2.5%.LC-MS analysis indicates the majority of the modification is outside ofthe CDR region and is at levels typical of other IgG4 antibodies. Threedegradation sites within the CDRs are identified changed less than 1%(pH 6; 4 wk at 25° C. minus 4° C. control). The lack of degradationsites within the CDRs is also consistent with no significant change inBIACore affinity or stoichiometry following four weeks at 40° C.incubation at either pH 4, 6, or 8.

C. Physical Stability

i) Freeze Thaw Stability

Antibody I is formulated under the following conditions:

-   -   a) 1 mg/mL in 10 mM Citrate, pH 6.0;    -   b) 1 mg/mL in 10 mM Citrate, pH 6.0, 0.02% Tween-80;    -   c) 1 or 50 mg/mL in 10 mM Citrate, pH 6.0, 150 mM NaCl; and    -   d) 1 or 50 mg/mL in 10 mM Citrate, pH 6.0, 150 mM NaCl, 0.02%        Tween-80.

These formulations are placed in a PC/min controlled freezing container(Nalgene, 5100-0001) and frozen in a −80° C. freezer for at least eighthours and then removed and thawed at room temperature for at least eighthours. This freeze/thaw cycle is repeated up to three times. Samples areremoved after one and three freeze thaw cycles and analyzed for HMWpolymer by SEC (see SEC method described in part A above) and insolubleparticle formation by HIAC particle counter (Pacific Scientific model9703 with low volume attachment). No significant increase in HMW polymerformation is observed following three freeze thaw cycles under anyconditions tested. For the 1 mg/ml formulations a significant increasein HIAC particle counts is observed only in the non-Tween-80 containingformulations. At 50 mg/ml particle counts were typical of other wellperforming IgG4 antibodies with particle counts (≧10 micron) wereapproximately 1500 counts/mL without Tween-80 and lowered toapproximately 280 with Tween-80.

ii) Static Hold at High Concentration

Antibody is formulation at 50 mg/mL under the following conditions:

-   -   a) 10 mM Citrate, pH 6.0, 150 mM NaCl; and    -   b) 10 mM Citrate, pH 6.0, 150 mM NaCl, 0.02% Tween-80

These formulations are held static for 4-weeks at 4 and 25° C. Thechange in HIAC particle counts (Pacific Scientific model 9703 with lowvolume attachment) is measured after 4-weeks at 25° C.

HIAC particle counts (≧10 micron) for Tween containing formulationsaverage 290 counts/mL (270 and 310) and moderately higher, 804 counts/mL(728 and 880) for formulations without Tween. These results are typicalof other IgG4 antibodies that exhibit good physical stability. These twoformulations were also stored in glass instead of standard plasticeppendorf tubes. Particle counts for the samples stored in glass are 4to 8 fold lower (average 35 and 191 counts/mL respective for with andwithout Tween).

SEQ ID Listing Heavy Chain CDRs SEQ ID NO: 1 GYKFTRYVMHSEQ ID NO: 2 YINPYNDGTNYNEKFKG SEQ ID NO: 3 ARNWDTGL Light Chain CDRsSEQ ID NO: 4 KASDHILKFLT SEQ ID NO: 5 GATSLET SEQ ID NO: 6 QMYWSTPFTHeavy Chain Variable Regions SEQ ID NO: 7 (Antibody I)QVQLVQSGAEVKKPGSSVKVSCKASGYKFTRYVMHWVRQAPGQGLEWMGYINPYNDGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNWDTGLWGQGTTVTVSSLight Chain Variable Regions SEQ ID NO: 8 (Antibody I)DIQMTQSPSSLSASVGDRVTITCKASDHILKFLTWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQMYWSTPFTFGGGTKVEIK Complete Heavy ChainSEQ ID NO: 9 (Antibody I)QVQLVQSGAEVKKPGSSVKVSCKASGYKFTRYVMHWVRQAPGQGLEWMGYINPYNDGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNWDTGLWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Complete Light ChainSEQ ID NO: 10 (Antibody I)DIQMTQSPSSLSASVGDRVTITCKASDHILKFLTWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQMYWSTPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Nucleotide SeuuencesHeavy Chain Variable Region SEQ ID NO: 11 (Antibody I)CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGATATAAATTCACTCGTTATGTTATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATATATTAATCCTTACAATGATGGTACTAACTACAATGAGAAGTTCAAAGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAACTGGGACACAGGCCTCTGGGGCCAAGGCACCACTGTCACAG TCTCCTCANucleotide Sequences Light Chain Variable RegionsSEQ ID NO: 12 (Antibody I)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAAGGCAAGTGACCACATTCTCAAATTTTTAACTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCAACCAGTTTGGAAACTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAATGTATTGGAGTACTCCGTTCACGTTCGGAGGGGGGACCAAGGTGGAAATAAAA Nucleotide SeouenceComplete Heavy Chain SEQ ID NO: 13 (Antibody I)CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGATATAAATTCACTCGTTATGTTATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATATATTAATCCTTACAATGATGGTACTAACTACAATGAGAAGTTCAAAGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAACTGGGACACAGGCCTCTGGGGCCAAGGCACCACTGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAAAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT Nucleotide Sequence Complete Light ChainSEQ ID NO: 14 (Antibody 1)GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAAGGCAAGTGACCACATTCTCAAATTTTTAACTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCAACCAGTTTGGAAACTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAATGTATTGGAGTACTCCGTTCACGTTCGGAGGGGGGACCAAGGTGGAAATAAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC Protein SequencesMature Human IL-23 p19 subunit amino acid sequence SEQ ID NO: 15RAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP

1. An antibody that binds to the p19 subunit of human IL-23 comprising alight chain and a heavy chain, wherein the light chain comprises a lightchain variable region (LCVR) and the heavy chain comprises a heavy chainvariable region (HCVR), wherein the LCVR comprises complementaritydetermining regions LCDR1, LCDR2, and LCDR3, and the HCVR comprisescomplementarity determining regions HCDR1, HCDR2, and HCDR3, whereinLCDR1 consists of amino acid sequence SEQ ID NO:4, LCDR2 consists ofamino acid sequence SEQ ID NO:5, LCDR3 consists of amino acid sequenceSEQ ID NO:6, HCDR1 consists of amino acid sequence SEQ ID NO:1, HCDR2consists of amino acid sequence SEQ ID NO:2, and HCDR3 consists of aminoacid sequence SEQ ID NO:3.
 2. An antibody that binds to the p19 subunitof human IL-23 comprising a light chain and a heavy chain, wherein thelight chain comprises a light chain variable region (LCVR) and the heavychain comprises a heavy chain variable region (HCVR), wherein the LCVRcomprises amino acid sequence SEQ ID NO: 8 and the HCVR comprises aminoacid sequence SEQ ID NO:
 7. 3. An antibody that binds to the p19 subunitof human IL-23 comprising a light chain and a heavy chain, wherein thelight chain comprises amino acid sequence SEQ ID NO: 10 and the heavychain comprises amino acid sequence SEQ ID NO:
 9. 4. An antibody thatbinds to the p19 subunit of human IL-23 comprising two light chains andtwo heavy chains, wherein each light chain comprises amino acid sequenceSEQ ID NO: 10 and each heavy chain comprises SEQ ID NO:
 9. 5. A DNAmolecule comprising a polynucleotide sequence encoding a light chainpolypeptide having the amino acid sequence SEQ ID NO:
 10. 6. A DNAmolecule comprising a polynucleotide sequence encoding a heavy chainpolypeptide having the amino acid sequence SEQ ID NO:
 9. 7. Arecombinant host cell comprising the DNA molecule of claim 5 and the DNAmolecule of claim 6, which cell is capable of expressing an antibodycomprising a heavy chain and a light chain, wherein the amino acidsequence of the heavy chain is SEQ ID NO: 9 and the amino acid sequenceof the light chain is SEQ ID NO:
 10. 8. A process for producing anantibody that binds to the p19 subunit of human IL-23 comprising a heavychain and a light chain, wherein the heavy chain comprises amino acidsequence SEQ ID NO: 9 and the light chain comprises the amino acidsequence of SEQ ID NO: 10, said process comprising the steps of: a)cultivating a recombinant host cell of claim 7, under conditions suchthat said antibody is expressed; and b) recovering from said host cellthe expressed antibody.
 9. An antibody produced by the process of claim8.
 10. A pharmaceutical composition comprising an antibody of claim 4and one or more pharmaceutically acceptable carriers, diluents orexcipients.
 11. A pharmaceutical composition comprising an antibody ofclaim 9 and one or more pharmaceutically acceptable carriers, diluentsor excipients.
 12. A method of treating or preventing an autoimmune orinflammatory condition in a patient, comprising administering to apatient in need thereof an effective amount of an antibody of claim 4,wherein the condition is selected from the group consisting of multiplesclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases,ankylosing spondylitis, graft-versus-host disease, lupus and metabolicsyndrome.
 13. A method of treating or preventing an autoimmune orinflammatory condition in a patient, comprising administering to apatient in need thereof an effective amount of an antibody of claim 9,wherein the condition is selected from the group consisting of multiplesclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases,ankylosing spondylitis, graft-versus-host disease, lupus and metabolicsyndrome.
 14. A method of treating or preventing cancer in a patient,comprising administering to a patient in need thereof an effectiveamount of an antibody of claim 4, wherein the cancer is melanoma, colon,ovarian, head and neck, lung, breast, or stomach cancer.
 15. A method oftreating or preventing cancer in a patient, comprising administering toa patient in need thereof an effective amount of an antibody of claim 9,wherein the cancer is melanoma, colon, ovarian, head and neck, lung,breast, or stomach cancer.